I. Field of the Invention
This invention relates to a method for increasing the yield of desired protein products obtainable by the expression of foreign genes in the baculovirus-cellular expression system using intermediate DNA modifications in the method, and to novel recombinant baculoviruses so-produced, particularly those which express HIV-1 specific rev, vif, pol and tat proteins upon infection of insect cells. The invention also relates to the utilization of these proteins for the development of prognostic reagents, diagnostic reagents and combined subunit vaccine against AIDS.
II. DESCRIPTION OF THE PRIOR ART
An important goal of recombinant DNA technology, as far as it relates to protein engineering, is to provide a gene expression system which will produce large quantities of desired gene products and provide protein modifications similar to those of the naturally occurring proteins.
Both prokaryotic and eukaryotic cells have been used to express cloned foreign genes and Escherichia coli is the most commonly used prokaryotic host system for foreign gene expression. However, prokaryotic cells are suitable for foreign gene expression only if the gene product does not require post-translational modifications such as glycosylation, phosphorylation or signal peptide cleavage. Since prokaryotic cells do not possess the appropriate machinery needed for the proper modification of many eukaryotic proteins, it has been necessary to develop gene expression systems using eukaryotes to obtain appropriately modified gene products There have been impressive successes in the expression of foreign genes using eukaryotic hosts such as yeast, mammalian, plant and insect cells. The impetus for the development of new systems has come mainly from the need to produce larger quantities of properly modified cloned gene products.
Advances in the genetics of invertebrate viruses and cells have allowed the development of viral-cellular systems which give both a high level of synthesis and complex processing of recombinant products. In particular, baculoviruses such as Autographa californica nucleopolyhedrosis virus (AcNPV) and Bombyx mori nucleopolyhedrosis virus (BmNPV) are extremely useful helper-independent eukaryotic expression vectors which are easily engineered. In the case of AcNPV, the system is based on a cell line established in the late 1970's from pural ovarian cells of the moth Spodoptera frugiperda. When infected with baculovirus carrying a foreign gene, these cells synthesize recombinant products complete with post translational modifications. In the case of BmNPV, foreign gene products can be expressed in living insects, namely silkworms. Both these viral systems are based on the utilization of the strong promoter of the gene encoding polyhedrin, the sole component of the crystalline matrix that acts as a protective shield for viral particles outside their insect host. The techniques conventionally employed in these systems are described in detail in U.S. Pat. No. 4,745,051 to Gale E. Smith et al issued on May 17, 1988; Baculovirus Vectors for Expression of Foreign Genes, C. Yong Kang, Advances in Virus Research, Vol. 35, pp 177-192, Academic Press Inc., 1988; A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Max D. Summers and Gale E. Smith, May 1987, Texas A & M University; and Baculoviruses as Gene Expression Vectors, Lois K. Miller, Ann. Rev. Microbiol. 42, pp 177-199, 1988; the disclosures of which are incorporated herein by reference. This expression system has been used for the successful production of large quantities of many different gene products including human fibroblast interferon, human c-mye protein, human interleukin 2, etc. However, not all genes under the polyhedrin gene promoter express at high levels, e.g. those for HIV-1 specific rev, vif, pol and tat, as mentioned above. Many researchers who are utilizing the baculovirus expression system have tried numerous techniques in order to improve the expression levels of such genes, but without much success (International Conference on Baculoviruses, Oxford, Great Britain, Aug. 30 Sep. 3, 1988). Accordingly, the products which can be successfully produced by the system to date have been dependent upon the control mechanism that nature has selected for high level expression.